Delivery systems for periadventitial delivery for treatment of restenosis and anastomotic intimal hyperplasia

ABSTRACT

The invention provides methods for treating injuries to one or more internal structures of a subject by administering a drug delivery vehicle to an external surface of the injured structure. The drug delivery vehicle substantially adheres to the site of administration and provides for the release of a bioactive agent that reduces or prevents further injury to the internal structure by disease processes, such as hyperplasia.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. patentapplication Ser. No. 60/178,087, filed on Jan. 25, 2000, the disclosureof which is incorporated herein in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] The hollow or tubular geometry of organs has functionalsignificance, such as in the facilitation of fluid or gas transport(e.g., blood, urine, lymph and respiratory gases) or cellularcontainment (e.g., sperm, ova). Disease processes may affect organs ortheir components by encroaching upon, obstructing or otherwise reducingthe cross-sectional area of the hollow or tubular elements. The abilityof the organ to properly function can be severely compromised. Anillustrative example of this phenomenon can be seen by reference tocoronary vasculature.

[0003] Coronary arteries are often subject to attack by diseaseprocesses, most commonly by atherosclerosis. In atherosclerosis, thecoronary vessels become lined with lesions known as plaques. Thedevelopment of plaques leads to a decrease in vessel cross-sectionalarea and a concomitant compromise in blood flow through the vessel. Thereduction in blood flow to the coronary muscle can result in clinicalangina, unstable angina or myocardial infarction and death.

[0004] Historically, the treatment of advanced atherosclerotic vasculardisease involved cardio-thoracic surgery in the form of coronary arterybypass grafting (CABG). Such artery bypass grafting is not limited touse with the coronary muscle, but is also used to treat heart and renalfailure, arterial aneurysms, and other conditions that require generalvascular bypass to restore blood flow to areas of ischemia. Anothercommonly used method for restoring blood flow to occluded vasculature ispercutaneous coronary angioplasty. Angioplasty is a routinely-utilizedsurgical procedure for the treatment of diseases, such asatherosclerosis and medial arteriosclerosis. Both CABG and angioplastynormally involve injury to a portion of an artery or vein. In many casesthe injury is followed by implantation of a donor or synthetic vasculargraft, stent, or other implant in order to replace or repair the injuredvascular or heart portion.

[0005] The treatment of intravascular diseases by angioplasty isrelatively non-invasive. Techniques, such as percutaneous transluminalangioplasty (PTA) and percutaneous transluminal coronary angioplasty(PTCA) typically involve use of a guide wire. A typical balloon catheterhas an elongate shaft with a balloon attached to its distal end and amanifold attached to the proximal end. In use, the balloon catheter isadvanced over the guide wire such that the balloon is positionedadjacent a restriction in a diseased vessel. The balloon is theninflated and the restriction in the vessel is dilated.

[0006] Vascular restrictions that have been dilated do not always remainopen. In up to 50% of the cases, a new restriction in the lumen of thevascular structure appears over a period of months. The newly formedrestriction, or “restenosis,” arises due to the onset and maintenance ofintimal hyperplasia at the site of insult. Restenosis and intimalhyperplasia following a procedure on a vascular structure is discussedin the following publications, see, for example Khanolkar, Indian HeartJ. 48:281-282 (1996); Ghannem et al., Ann. Cardiol. Angeiol. 45:287-290(1996); Macander et al., Cathet. Cardiovasc. Diagn. 32:125-131; Strausset al., J. Am. Coll. Cardiol. 20:1465-1473 (1992); Bowerman et al.,Cathet. Cardiovasc. Diagn. 24:248-251 (1991); Moris et al., Am. Heart.J. 131:834-836 (1996); Schomig et al., J. Am. Coll. Cardiol.23:1053-1060 (1994); Gordon et al., J. Am. Coll. Cardiol. 21:1166-1174;and Baim et al., Am. J. Cardiol. 71:364-366 (1993).

[0007] Intimal hyperplasia also arises in conjunction with vascularreconstructive surgery. Vascular reconstructive surgery involvesremoving or reinforcing an area of diseased vasculature. Followingremoval of the diseased portion of the vessel, a prosthetic device, suchas an endovascular stent graft or prosthetic graft is implanted at thesite of removal. The graft is typically a segment of autologous orheterologous vasculature or, alternatively, it is a synthetic devicefabricated from a polymeric material. Stent grafts are generallyfabricated from metals, polymers and combinations of these materials.Similar to the situation with angioplasty, intimal hyperplasia alsocauses failure of implanted prosthetics in vascular reconstructivesurgery. Thus, a method to reduce the failure rate for angioplasty andvascular reconstructive surgery by preventing or reducing intimalhyperplasia is an avidly sought goal.

[0008] Intimal hyperplasia is the result of a complex series ofbiological processes initiated by vascular injury followed by plateletaggregation and thrombus formation with a final pathway of smooth musclecell migration and proliferation and extracellular matrix deposition.Platelets adhere and aggregate at the site of injury and releasebiologically active substances, the most important of which areplatelet-derived growth factors (Scharf et al., Blut 55:1131-1144(1987)). It has been postulated that intimal hyperplasia production isdriven by two principal mechanisms; platelet activation with the releaseof platelet-derived growth factors, and activation of the coagulationcascade with thrombus formation, which also results in the release ofbiologically active substances, which can contribute to smooth musclecell proliferation (Chervu et al., Surg. Gynecol. Obstet. 171:433-447,1990)).

[0009] Attempts to prevent the onset, or to mitigate the effects, ofintimal hyperplasia have included, for example, drug therapy withantihyperplastic agents, such as antiplatelet agents (e.g. aspirin,arachidonic acid, prostacyclin), antibodies to platelet-derived growthfactors, and antithrombotic agents (e.g. heparin, low molecular weightheparins) (see, Ragosta et al. Circulation 89: 11262-127 (1994)).Clinical trials using antihyperplastic agents, however, have shownlittle effect on the rate of restenosis (Schwartz, et al., N. Engl. J.Med. 318:1714-1719, (1988); Meier, Eur. Heart J. 10 (suppl G):64-68(1989)). In both angioplasty and vascular reconstructive surgery, druginfusion near the site of stenosis has been proposed as a means toinhibit restenosis. For example, U.S. Pat. No. 5,558,642 to Schweich etal. describes drug delivery devices and methods for deliveringpharmacological agents to vessel walls in conjunction with angioplasty.

[0010] In addition to simply administering a bioactive agent to apatient to prevent restenosis, a number of more sophisticated methodshave been investigated. For example, to address the restenosis problemin vascular reconstruction, it has been proposed to provide stents whichare seeded with endothelial cells (Dichek et al., Circulation80:1347-1353(1989). Both autologous and heterologous cells have beenused (see, for example, Williams, U.S. Pat. No. 5,131,907, which issuedon Jul. 21, 1992; and Herring, Surgery 84:498-504 (1978)). Methods ofproviding therapeutic substances to the vascular wall by means ofdrug-coated stents have also been proposed. For example, methotrexateand heparin have been incorporated into a cellulose ester stent coating.The drug treated stent, however, failed to show a reduction inrestenosis when implanted in porcine coronary arteries (Cox et al.,Circulation 84: II71 (1991)). Implanted stents have also been used tocarry thrombolytic agents. For example, U.S. Pat. No. 5,163,952 to Froixdiscloses a thermal memoried expanding plastic stent device, which canbe formulated to carry a medicinal agent by utilizing the material ofthe stent itself as an inert polymeric drug carrier. Pinchuk, in U.S.Pat. No. 5,092,877, discloses a stent of a polymeric material which canbe employed with a coating that provides for the delivery of drugs. Dinget al., U.S. Pat. No. 5,837,313 disclose a method of coating animplantable open lattice metallic stent prosthesis with a drug releasingcoating.

[0011] Other patents which are directed to devices of the classutilizing biodegradable or biosorbable polymers include, for example,Tang et al., U.S. Pat. No. 4,916,193, and MacGregor, U.S. Pat. No.4,994,071. Sahatjian in U.S. Pat. No. 5,304,121, discloses a coatingapplied to a stent consisting of a hydrogel polymer and a preselecteddrug; possible drugs include cell growth inhibitors and heparin. Drugshave also been delivered to the interior of vascular structures by meansof a polyurethane coating on a stent. The coating was swelled and abiologically active compound was incorporated within the interstices ofthe polymer (Lambert, U.S. Pat. No. 5,900,246, which issued May 4,1999).

[0012] The use of stents, as described above, is accompanied by certaindisadvantages. For example, in many cases, it is desirable toprecondition the structure with anti-hyperplastic agents prior to theirundergoing a surgical procedure. As placing a stent requires disruptingthe border of the structure in which the stent is to be placed, it isnot possible to use a drug-coated stent to precondition a tissue.Moreover, when the drug has diffused out of a drug-loaded stent, it isnot possible to administer additional doses of the drug if necessarywithout replacing the stent and subjecting the repaired structure toadditional trauma.

[0013] In another method, Edelman et al. have utilized a solid matrix,seeded with vascular endothelial cells (U.S. Pat. No. 5,766,584). Thedelivery vehicle consists of a three-dimensional matrix onto whichendothelial cells are seeded. When the seeded endothelial cells havereached the desired density within the matrix, a vascular structure thathas undergone an invasive procedure is wrapped with the seeded matrix.The endothelial cells within the matrix secrete products that diffuseinto the surrounding tissue without migrating to the endothelial celllining of the blood vessel. A procedure that relies on wrapping aninjured vascular structure with a delivery matrix is less than ideal.For example, as it is generally desirable for the surgical procedure tobe minimally invasive and for the surgical field to be of the smallestpossible size, there is a stringent practical limitation the size of thearea that can be wrapped and the thickness of the matrix wrapped aroundthe circumference of a vascular structure. Moreover, the endothelialcell-based approaches have not been broadly accepted, because theyrequire that endothelial cell cultures from a patient be established andthat the cells be seeded at high densities within the polymeric matrix.

[0014] In another method, a modulator of cell or tissue growth isdelivered to an extraluminal site adjacent to the point of vascularinjury by means of an implanted infusion pump or biodegradable vehicle(Edelman, et al., U.S. Pat. No. 5,527,532). In one embodiment of theEdelman invention, the biodegradable vehicle is implanted in theadventitia at a site adjacent to the site of injury. The modulator isdelivered to the adventitia and from the aventitia to exterior surfaceof the vascular wall.

[0015] Neither of the methods disclosed by Edelman et al. addresscoating directly the exterior surface of a vascular or other tubularstructure with a flowable drug delivery matrix into which a therapeuticagent has been dispersed. Moreover, Edelman et al. does not disclose theuse of a delivery vehicle that is substantially adherent to the exteriorsurface of an internal structure of a patient.

[0016] A method of preventing or retarding intimal hyperplasia bydelivering a therapeutic agent to the site of injury using a drugdelivery vehicle implanted on the exterior surface of the injuredstructure would represent a substantial advance in the art. Moreover, itwould be desirable if the method was flexible enough to allow the agentsto be applied prior to the surgery and to be reapplied following thesurgery. Quite surprisingly, the present invention provides such amethod.

SUMMARY OF THE INVENTION

[0017] It has now been discovered that antihyperplastic and other usefulagents can be delivered to internal organs and other tissues by aperiadventitial route by layering a bioadhesive material containing adesired agent on the exterior surface of the organ or other tissue.Although the methods described herein are of general applicability, thepresent invention particularly concerns a method for inhibiting intimalhyperplasia induced by arterial interventions by administering,periadventitially at the site of the vascular injury, a bioactivecompound that inhibits intimal hyperplasia.

[0018] Thus, in a first aspect, the present invention provides a methodof preventing or reducing intimal hyperplasia at a site of insult to aninternal structure in a subject. The method comprises, contacting anexterior surface of the internal structure with a drug delivery vehicle.The drug delivery vehicle is generally deposited as a substantiallyflowable liquid or semi-liquid material onto the exterior surface of theinternal structure to which it will, preferably, substantially adhere.The drug delivery vehicle comprises at least one intimalhyperplasia-preventing agent that is released from the drug deliveryvehicle in a time dependent manner and in an amount effective to preventor reduce intimal hyperplasia.

[0019] In a second aspect, the present invention provides a method ofpreventing or reducing intimal hyperplasia at a site of insult to avascular structure in a subject. The insult is a member selected fromthe group consisting of angioplasty, vascular reconstructive surgery andcombinations thereof. The method comprises, contacting an exteriorsurface of the internal structure with a drug delivery vehicle. The drugdelivery vehicle is generally deposited as a substantially flowableliquid or semi-liquid material onto the exterior surface of the internalstructure to which it will, preferably, substantially adhere. The drugdelivery vehicle comprises at least one intimal hyperplasia-preventingagent that is released from the drug delivery vehicle in a timedependent manner and in an amount effective to prevent or reduce intimalhyperplasia.

[0020] In a third aspect, the present invention provides a method oftreating a disease state of an internal structure in a subject. Themethod comprises surgically treating the disease state. The surgicaltreatment creates a surgical site that is treated by contacting anexterior surface of the internal structure with a drug delivery vehicle.The region of the external surface contacted with the drug deliveryvehicle is contiguous with the surgical site. The drug delivery vehicleis deposited on the external surface as a substantially flowable liquidor semi-liquid material. The drug delivery vehicle is, preferably,substantially adherent to the external surface of the internalstructure. The drug delivery vehicle comprises one or more intimalhyperplasia-preventing agent that is released in a time dependent mannerand in an amount effective to prevent or reduce said intimalhyperplasia.

[0021] Other objects and advantages of the present invention will beapparent from the detailed description that follows

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

[0022] A. Definitions

[0023] As used herein, the term “contiguous,” refers to a location thatis coextensive with the site of a surgical or other insult. When theinsult is angioplasty, an area “contiguous” with the site of insult willgenerally be on an area of the external surface of the vascularstructure.

[0024] “Internal structure,” as used herein refers to structures such asvascular structures (e.g., vessels, heart), organs (e.g., stomach,liver, intestines) and the like. Preferred internal structures includethose having a substantially circular cross-section, such as the organsof the digestive system and reproductive and urinary systems.

[0025] “Time dependent manner,” as used herein refers to the release ofa drug from a drug delivery vehicle with zero-order or higher orderkinetics.

[0026] “Flowable,” as used herein refers to the ability to extrude thevehicle through an opening in a delivery device, such as a needle,catheter, atomizer and the like.

[0027] B. Introduction

[0028] Intimal hyperplasia (also referred to as neointimal hyperplasia)refers to a proliferative response to a vascular injury consisting ofsmooth muscle cells (SMCs) which form an intimal lesion on the luminalsurface around the inner circumference of a blood vessel (intima)following a vascular intervention such as, e.g., angioplasty orendarterectomy. The hyperplastic growth gradually encroaching into thelumen of the blood vessel is the leading cause of restenosis.Hyperplasia occurs gradually over a period of days to several weeksfollowing the arterial intervention, as distinguished from a thrombus,such as may occur in the circulating blood immediately at the time ofintervention.

[0029] It is estimated that well over one million arterial interventionsare performed each year in the United States for the treatment ofocclusive arterial disease (see, Califf et al., J. Am. Coll. Cardiol.17:2B-13B (1991)). The early results of these procedures are generallyexcellent. Within about six months to five years, however, over 50% ofthe treated arteries develop restenosis and require reintervention. Mostdevelop restenosis within the first year. Consequently, in many clinics,up to 50% of the case load consists of secondary procedures as opposedto first interventions.

[0030] Thus, in a first aspect, the present invention provides a methodof preventing or reducing intimal hyperplasia at a site of insult to aninternal structure in a subject. The method comprises, contacting anexterior surface of the internal structure with a drug delivery vehicle.The drug delivery vehicle is generally deposited as a substantiallyflowable liquid or semi-liquid material onto the exterior surface of theinternal structure to which it will, preferably, substantially adhere.The drug delivery vehicle comprises at least one intimalhyperplasia-preventing agent that is released from the drug deliveryvehicle in a time dependent manner and in an amount effective to preventor reduce intimal hyperplasia.

[0031] In another aspect, the present invention provides a method oftreating a disease state of an internal structure in a subject. Themethod comprises surgically treating the disease state. The surgicaltreatment creates a surgical site that is treated by contacting anexterior surface of the internal structure with a drug delivery vehicle.The region of the external surface contacted with the drug deliveryvehicle is contiguous with the surgical site. The drug delivery vehicleis deposited onto the external surface of the of the internal structureas a flowable liquid or semi-liquid material. The depositeddrug-delivery matrix will, preferably, substantially adhere to theexternal surface of the internal structure. The drug delivery vehiclecomprises one or more intimal hyperplasia preventing agent that isreleased in a time dependent manner and in an amount effective toprevent or reduce said intimal hyperplasia.

[0032] The method of the present invention can be practiced on anyinternal structure of any mammal. In a presently preferred embodiment,the internal structure is a structure having a substantially circularcross-section. Exemplary structures having substantially circularcross-sections include, but are not limited to, vascular systemcomponents, intestinal system components, urinary system components,reproductive system components and combinations thereof. In a presentlypreferred embodiment, the internal structure is a vascular structure.

[0033] The method of the invention can be practiced in conjunction withsubstantially to an internal structure, including, for example, adisease, a degenerative condition, an injury or trauma, and a surgicalinsult.

[0034] In a preferred embodiment, the insult is a surgical insult. In afurther preferred embodiment, the surgical insult derives from atechnique, such as angioplasty, vascular reconstructive surgery, heartvalve replacement, heart transplantation and combinations thereof. In apresently preferred embodiment, the method is practiced in conjunctionwith vascular reconstructive surgery, angioplasty and combinationsthereof.

[0035] In another preferred embodiment, the surgical injury comprisesplacing a prosthesis at the site of insult to the internal structure.Preferred prostheses include, but are not limited to stents, grafts,valves or a combination thereof. When a prosthetic device is implanted,the method of the invention is preferably practiced by contacting theprosthetic, the site of insult and combinations thereof with the drugdelivery vehicle. In a preferred embodiment, the drug delivery vehicleis layered on the exterior surface of the internal structure so that thedelivery vehicle encompasses both the prosthesis and the site of insult.

[0036] In another preferred embodiment, the insult is, for example, CABGand the site of insult comprises an anastomosis. In this embodiment, theexterior surface of the vascular structure contacted with the drugdelivery vehicle comprises the anastomosis.

[0037] The method of the invention can be practiced at substantially anytime relative to the onset of the insult. For example, in a preferredembodiment, the insult is a surgical insult and the method is practicedon an internal structure prior to its undergoing the surgical insult asa form of presurgical conditioning. In this embodiment, the method canbe practiced again during and/or following the surgical insult. Inanother preferred embodiment, the method is practiced during surgery andis optionally practiced one or more times following surgery. Otherappropriate times relative to insult for practicing the method of theinvention will be apparent to those of skill in the art.

[0038] C. Bioactive Agents

[0039] Any bioactive agent that is capable of retarding or arresting theformation of intimal hyperplasia is appropriate for incorporation intothe coating of the invention. For reasons of clarity, the discussionthat follows is focused on vascular reconstructive surgery involvingimplanting a vascular graft. Those of skill will readily appreciate thatthe discussion is generally applicable to other forms of vascularreconstructive surgery, angioplasty and preventing the formation ofpost-surgical adhesions in other organs and/or internal structures.

[0040] Intimal hyperplasia is caused by a cascade of events in responseto vascular damage. As part of the inflammatory and reparative responseto vascular damage, such as that resulting from vascular surgeries,inflammatory cells (e.g., monocytes, macrophages, and activatedpolymorphonuclear leukocytes and lymphocytes) often form inflammatorylesions in the blood vessel wall. Lesion formation activates cells inthe intimal and medial cellular layers of the blood vessel or heart. Thecellular activation may include the migration of cells to the innermostcellular layers, known as the intima. Such migrations pose a problem forthe long-term success of vascular grafts because endothelial cellsrelease smooth muscle cell growth factors (e.g., platelet-derived growthfactor, interleukin-1, tumor necrosis factor, transforming growthfactor-beta, and basic fibroblast growth factor), that cause thesenewly-migrated smooth muscle cells to proliferate. Additionally,thrombin has been demonstrated to promote smooth muscle cellproliferation both by acting as a growth factor itself and by enhancingthe release of several other growth factors produced by platelets andendothelial cells (Wu et al., Annu. Rev. Med. 47:315-31 (1996)). Smoothmuscle cell proliferation causes irregular and uncontrolled growth ofthe intima into the lumen of the blood vessel or heart, which constrictsand often closes the vascular passage. Often, irregular calcium depositsin the media or lipid deposits in the intima accompany smooth musclecell growths, such lipid deposits normally existing in the form ofcholesterol and cholesteryl esters that are accumulated withinmacrophages, T lymphocytes, and smooth muscle cells. These calcium andlipid deposits cause arteriosclerotic hardening of the arteries andveins and eventual vascular failure. These arteriosclerotic lesionscaused by vascular grafting can also be removed by additionalreconstructive vascular surgery, but the failure rate of this approachdue to restenosis has been observed to be between thirty and fiftypercent.

[0041] Any bioactive agent that can interrupt or retard one or more ofthe elements of the above-described hyperplastic cascade is useful inpracticing the present invention. Example of useful bioactive agentsinclude, but are not limited to, antithrombotics, antiinflammatories,corticosteroids, antimicrotubule agents, antisense oligonucleotides,antineoplastics, antioxidants, antiplatelets, calcium channel blockers,converting enzyme inhibitors, cytokine inhibitors, growth factors,growth factor inhibitors, growth factor sequestering agents,immunosuppressives, tissue factor inhibitor, smooth muscle inhibitors,organoselenium compounds, retinoic acid, retinoid compounds, sulfatedproteoglycans, superoxide dismutase mimics, NO, NO precursors andcombinations thereof.

[0042] Certain biologically active agents falling within theabove-recited classes are presently preferred. For example, when one ormore of the bioactive agents is an antithrombotic agent, it ispreferably selected from heparin, hirudin or a combination thereof. Whenone or more of the bioactive agents is a corticosteriod, it ispreferably selected from dexamethasone, a dexamethosone derivative or acombination thereof. When one or more of the bioactive agents is anantimicrotubule agent, it is preferably selected from taxane, aderivative of taxane or a combination thereof. When one or more of thebioactive agents is an antiplatelet agent, the agent is preferably aninhibitor of collagen synthesis, such as halofuginore, derivatives ofhalofuginore, proteins (e.g., GpII_(b)III_(a), ReoPro™) or a combinationthereof.

[0043] Pharmaceutically acceptable salts of the biologically activeagents are also of use in the present invention. Exemplary salts includethe conventional non-toxic salts of the compounds of this invention asformed, e.g., from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,trifluoroacetic and the like.

[0044] Other agents that are useful in conjunction with the presentinvention will be readily apparent to those of skill in the art.

[0045] D. Incorporation of Bioactive Agents

[0046] The bioactive agents useful in practicing the present inventioncan be incorporated into drug delivery vehicles (“coatings”) useful inpracticing the methods of the invention using one or more of the manyart-recognized techniques for immobilizing, or adhering, drug moleculesto other molecules and surfaces. These methods include, but are notlimited to, covalent attachment to the coating of the drug or aderivative of the drug bearing a “handle” allowing it to react with acomponent of the delivery vehicle having a complementary reactivity.Moreover, the bioactive agent can be incorporated into the vehicle usinga non-covalent interaction, such as an electrostatic or an ionicattraction between a charged drug and a component of the coating bearinga complementary charge. The bioactive agents can also be admixed, andnot otherwise interact with, the components of the delivery vehicle. Thecoatings can also be fabricated to incorporate the drugs into reservoirslocated in the coating. The reservoirs can have a variety of shapes,sizes and they can be produced by an array of methods. For example, thereservoir can be a monolithic structure located in one or morecomponents of the coating. Alternatively, the reservoir can be made upof numerous small microcapsules that are, for example, embedded in thematerial from which the coating is fabricated. Furthermore, thereservoir can be a coating that includes the bioactive agent diffusedthroughout, or within a portion, of the coating's three-dimensionalstructure. The reservoirs can be porous structures that allow the drugto be slowly released from its encapsulation, or the reservoir caninclude a material that bioerodes following implantation and allows thedrug to be released in a controlled fashion.

[0047] 1. Covalently Attached Bioactive Materials

[0048] In a preferred embodiment, the biologically active material iscovalently bonded to a reactive group located on one or more componentsof the coating. The art is replete with methods for preparingderivatized, polymerizable monomers, attaching bioactive materials ontopolymeric surfaces and derivatizing bioactive materials and polymers toallow for this attachment (see, for example, Hermanson, BIOCONJUGATETECHNIQUES, Academic Press, 1996, and references therein). Commonapproaches include the use of coupling agents such as glutaraldehyde,cyanogen bromide, p-benzoquinone, succinic anhydrides, carbodiimides,diisocyanates, ethyl chloroformate, dipyridyl disulfide,epichlorohydrin, azides, among others, which serve as attachmentvehicles for coupling reactive groups of biologically active moleculesto reactive groups on a monomer or a polymer.

[0049] A polymer can be functionalized with reactive groups by, forexample, including a moiety bearing a reactive group as an additive to ablend during manufacture of the polymer or polymer precursor. Theadditive is dispersed throughout the polymer matrix, but does not forman integral part of the polymeric backbone. In this embodiment, thesurface of the polymeric material is altered or manipulated by thechoice of additive or modifier characteristics. The reactive groups ofthe additive are used to bind one or more bioactive agents to thepolymer.

[0050] A useful method of preparing surface-functionalized polymericmaterials by this method is set forth in, for example, Caldwell, U.S.Pat. No. 5,874,164, issued Feb. 23, 1999. In the Caldwell method,additives or modifiers are combined with the polymeric material duringits manufacture. These additives or modifiers include compounds thathave reactive sites, compounds that facilitate the controlled release ofagents from the polymeric material into the surrounding environment,catalysts, compounds that promote adhesion between the bioactivematerials and the polymeric material and compounds that alter thesurface chemistry of the polymeric material.

[0051] In another embodiment, polymerizable monomers bearing reactivegroups are incorporated in the polymerization mixture. Thefunctionalized monomers form part of the polymeric backbone and,preferably, present their reactive groups on the surface of the polymer.

[0052] Reactive groups contemplated in the practice of the presentinvention include functional groups, such as hydroxyl, carboxyl,carboxylic acid, amine groups, and the like, that promote physicaland/or chemical interaction with the bioactive material. The particularcompound employed as the modifier will depend on the chemicalfunctionality of the biologically active agent and can readily bededuced by one of skill in the art. In the present embodiment, thereactive site binds a bioactive agent by covalent means. It will,however, be apparent to those of skill in the art that these reactivegroups can also be used to adhere bioactive agents to the polymer byhydrophobic/hydrophilic, ionic and other non-covalent mechanisms.

[0053] In addition to manipulating the composition and structure of thepolymer during manufacture, a preferred polymer can also be modifiedusing a surface derivitization technique. There are a number ofsurface-derivatization techniques appropriate for use in fabricating thedelivery vehicles of the present invention (e.g., grafting techniques).These techniques for creating functionalized polymeric surfaces are wellknown to those skilled in the art. For example, techniques based onceric ion initiation, ozone exposure, corona discharge, UV irradiationand ionizing radiation (⁶⁰Co, X-rays, high energy electrons, plasma gasdischarge) are known and can be used in the practice of the presentinvention.

[0054] Substantially any reactive group that can be reacted with acomplementary component on a biologically active material can beincorporated into a polymer and used to covalently attach thebiologically active material to the coating of use in the invention. Ina preferred embodiment, the reactive group is selected fromamine-containing groups, hydroxyl groups, carboxyl groups, carbonylgroups, and combinations thereof. In a further preferred embodiment, thereactive group is an amino group.

[0055] Aminated polymeric materials useful in practicing the presentinvention can be readily produced through a number of methods well knownin the art. For example, amines may be introduced into a preformedpolymer by plasma treatment of materials with ammonia gas as found inHolmes and Schwartz, Composites Science and Technology, 38: 1-21 (1990).Alternatively, amines can be provided by grafting acrylamide to thepolymer followed by chemical modification to introduce amine moieties bymethods well known to those skilled in the art, e.g., Hofmannrearrangement reaction. A grafted acrylamide-containing polymer may beprepared by radiation grafting as set forth in U.S. Pat. No. 3,826,678to Hoffman et al. A grafted N-(3-aminopropyl)methacrylamide-containingpolymer may be prepared by ceric ion grafting as set forth in U.S. Pat.No. 5,344,455 to Keogh et al., which issued on Sep. 6, 1994.Polyvinylamines or polyalkylimines can also be covalently attached topolyurethane surfaces according to the method taught by U.S. Pat. No.4,521,564 to Solomone et al., which issued on Jun. 5, 1984.Alternatively, for example, aminosilane may be attached to the surfaceas set forth in U.S. Pat. No. 5,053,048 to Pinchuk, which issued on Oct.1, 1991.

[0056] In an exemplary embodiment, a polymeric coating material, or aprecursor material is exposed to a high frequency plasma with microwavesor, alternatively, to a high frequency plasma combined with magneticfield support to yield the desired reactive surfaces bearing at least asubstantial portion of reactant amino groups upon the substrate to bederivatized with the bioactive material.

[0057] A functionalized coating surface can also be prepared by, forexample, first submitting a coating component to a chemical oxidationstep. This chemical oxidation step is then followed, for example, byexposing the oxidized substrate to one or more plasma gases containingammonia and/or organic amine(s) which react with the treated surface.

[0058] In a preferred embodiment, the gas is selected from the groupconsisting of ammonia, organic amines, nitrous oxide, nitrogen, andcombinations thereof. The nitrogen-containing moieties derived from thisgas are preferably selected from amino groups, amido groups, urethanegroups, urea groups, and combinations thereof, more preferably primaryamino groups, secondary amino groups, and combinations thereof.

[0059] In another preferred embodiment, the nitrogen source is anorganic amine. Examples of suitable organic amines include, but are notlimited to, methylamine, dimethylamine, ethylamine, diethylamine,ethylmethylamine, n-propylamine, allylamine, isopropylamine,n-butylamine, n-butylmethylamine, n-amylamine, n-hexylamine,2-ethylhexylamine, ethylenediamine, 1,4-butanediamine,1,6-hexanediamine, cyclohexylamine, n-methylcyclohexylamine,ethyleneimine, and the like.

[0060] In further preferred embodiment, the chemical oxidation step issupplemented with, or replaced by, submitting the polymer to one or moreexposures to plasma-gas that contains oxygen. In yet a further preferredembodiment, the oxygen-containing plasma gas further contains argon (Ar)gas to generate free radicals. Immediately after a first-step plasmatreatment with oxygen-containing gases, or oxygen/argon plasma gascombinations, the oxidized polymer is preferably functionalized withamine groups. As mentioned above, functionalization with amines can beperformed with plasma gases such as ammonia, volatile organic amines, ormixtures thereof.

[0061] In an exemplary embodiment utilizing ammonia and/or organicamines, or mixtures thereof, as the plasma gases, a frequency in theradio frequency (RF) range of from about 13.0 MHz to about 14.0 MHz isused. A generating power of from 0.1 Watts per square centimeter toabout 0.5 Watts per square centimeter of surface area of the electrodesof the plasma apparatus is preferably utilized. An exemplary plasmatreatment includes evacuating the plasma reaction chamber to a desiredbase pressure of from about 10 to about 50 mTorr. After the chamber isstabilized to a desired working pressure, ammonia and/or organic aminegases are introduced into the chamber. Preferred flow rates aretypically from about 200 to about 650 standard mL per minute. Typicalgas pressure ranges from about 0.01 to about 0.5 Torr, and preferablyfrom about 0.2 to about 0.4 Torr. A current having the desired frequencyand level of power is supplied by means of electrodes from a suitableexternal power source. Power output is up to about 500 Watts, preferablyfrom about 100 to about 400 Watts. The plasma treatment can be performedby means of a continuous or batch process.

[0062] In the case of batch plasma treatment, a preferred plasma surfacetreatment system is the PLASMA SCIENCE PS 0350 (HIMONT/PLASMA SCIENCE,Foster City, Calif.)

[0063] Optimization procedures for the plasma treatment and the effectof these procedures on the characteristics and the performance of thereactive polymers can be determined by, for example, evaluating theextent of substrate functionalization. Methods for characterizingfunctionalized polymers are well known in the art.

[0064] The result of the above-described exemplary methods is preferablya polymeric surface, which contains a significant number of primaryand/or secondary amino groups. These groups are preferably readilyreactive at room temperature with an inherent, or an appended, reactivefunctional group on the bioactive material.

[0065] Once the amine-containing polymeric coating is prepared, it canbe used to covalently bind biologically active molecules having avariety of functional groups including, for example, ketones, aldehydes,activated carboxyl groups (e.g. activated esters), alkyl halides and thelike.

[0066] Synthesis of specific biologically active material-polymerconjugates is generally accomplished by: 1) providing a coatingcomponent comprising an activated polymer, such as an acrylic acid, anda biologically active agent having a position thereon which will allow alinkage to form; 2) reacting the complementary substituents of thebiologically active agent and the coating component in an inert solvent,such as methylene chloride, chloroform or DMF, in the presence of acoupling reagent, such as 1,3-diisopropylcarbodiimide or any suitabledialkyl carbodiimide (Sigma Chemical), and a base, such asdimethylaminopyridine, diisopropyl ethylamine, pyridine, triethylamine,etc. Alternative specific syntheses are readily accessible to those ofskill in the art (see, for example, Greenwald et al., U.S. Pat. No.5,880,131, issued Mar. 9, 1999.

[0067] By way of example, the discussion below is concerned with theattachment of a peptide-based bioactive material to an amine-containingpolymeric component of a coating of use in practicing the methods of theinvention. The choice of a peptide-based biologically active materialand an amine-containing polymer is intended to be illustrative of theinvention and does not define its scope. It will be apparent to those ofskill in the art how to attach a wide range of biologically activeagents to polymers comprising amines and other reactive groups.

[0068] The conjugates of use in practicing the instant invention, whichcomprise a peptide, can be synthesized by techniques well known in themedicinal chemistry art. For example, a free amine moiety on a polymericcoating component can be covalently attached to an oligopeptide at thecarboxyl terminus such that an amide bond is formed. Similarly, an amidebond may be formed by covalently coupling an amine moiety of anoligopeptide and a carboxyl moiety of a polymeric coating component. Forthese purposes, a reagent such as2-(1H-benzotriazol-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate(known as HBTU) and 1-hyroxybenzotriazole hydrate (known as HOBT),dicyclohexylcarbodiimide (DCC),N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC),diphenylphosphorylazide (DPPA),benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate (BOP) and the like, in combination, or singularly,can be utilized.

[0069] Furthermore, the instant conjugate can be formed by anon-peptidyl bond between a peptide and a coating component. Forexample, a peptide can be attached to a coating component through acarboxyl terminus of an oligopeptide via a hydroxyl moiety on apolymeric coating component, thereby forming an ester linkage. For thispurpose, a reagent such as a combination of HBTU and HOBT, a combinationof BOP and imidazole, a combination of DCC and DMAP, and the like can beutilized.

[0070] The instant conjugate can also be formed by attaching theoligopeptide to the polymeric coating component using a linker unit.Such linker units include, for example, a biscarbonyl alkyl diradicalwhereby an amine moiety on the coating component is connected with thelinker unit to form an amide bond and the amino terminus of theoligopeptide is connected with the other end of the linker unit alsoforming an amide bond. Conversely, a diaminoalkyl diradical linker unit,whereby a carbonyl moiety on the coating component is covalentlyattached to one of the amines of the linker unit while the other amineof the linker unit is covalently attached to the C-terminus of theoligopeptide, can also be utilized. Other such linker units, which arestable to the physiological environment, are also envisioned.

[0071] In addition to linkers that are stable in vivo, linkers that aredesigned to be cleaved to release the biologically active agent from thepolymer are useful in the methods of the present invention. Many suchlinker arms are accessible to those of skill in the art. Commoncleavable linker arms include, for example, specific protease cleavagesequences, disulfides, esters and the like. Many appropriate cleavablecross-linking agents are commercially available from companies, such asPierce (Rockford, Ill.), or can be prepared by art-recognized methods.

[0072] Any of the bioactive agents from the various classes of bioactiveagents set forth above can be tethered to a polymer by the methodsdescribed herein. In a particularly preferred embodiment, thebiologically active material is a taxane. For purposes of the presentinvention, the term “taxane” includes all compounds within the taxanefamily of terpenes. Thus, taxol (paclitaxel), 3′-substitutedtert-butoxy-carbonyl-amine derivatives (taxoteres) and the like as wellas other analogs available from, for example, Sigma Chemical (St. Louis,Mo.) and/or Bristol Meyers Squibb are within the scope of the presentinvention.

[0073] Generally, it is preferred that a taxane having the 2′ positionavailable for substitution is reacted with a suitably activated polymersuch as a polymeric carboxylic acid under conditions sufficient to causethe formation of a 2′ ester linkage between the two substituents.

[0074] One skilled in the art understands that in the synthesis ofcompounds useful in practicing the present invention, one may need toprotect various reactive functionalities on the starting compounds andintermediates while a desired reaction is carried out on other portionsof the molecule. After the desired reactions are complete, or at anydesired time, normally such protecting groups will be removed by, forexample, hydrolytic or hydrogenolytic means. Such protection anddeprotection steps are conventional in organic chemistry. One skilled inthe art is referred to PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, McOmie,ed., Plenum Press, NY, N.Y. (1973); and, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Greene, ed., John Wiley & Sons, NY (1981) for the teaching ofprotective groups which may be useful in the preparation of compounds ofthe present invention.

[0075] 2. Reversibly Associated Bioactive Materials

[0076] Generally, if it is desired that the biologically active agentremain active in the coating for a long period of time, it is preferableto covalently attach the biologically active molecule to the coatingitself. In an exemplary embodiment, a bioactive agent is immobilized ona component (e.g., fibrin) of a fibrin sealant. In contrast, if it isdesired that the biologically active agent escape the coating (e.g., bydiffusion from the coating, erosion of the coating, etc.), the agentshould be reversibly associated with the coating. The reversiblyassociated agent can, for example, be entrapped in a delivery matrix byadding the agent to the matrix components during manufacture of thematrix. In an exemplary embodiment, the agent is added to a polymer meltor a solution of the polymer. Other methods for reversibly incorporatingagents into a delivery matrix will be apparent to those of skill in theart.

[0077] Examples of such reversible associations include, for example,agents that are mechanically entrapped within the matrix and agents thatare encapsulated in structures (e.g., within microspheres, liposomes,etc.) that are themselves entrapped in, or immobilized on, the matrix.Other reversible associations include, but are not limited to, agentsthat are adventitiously adhered to the coating by, for example,hydrophobic or ionic interactions and agents bound to one or morecoating component by means of a linker cleaved by one or morebiologically relevant process. The reversibly associated agents can beexposed on the coating surface or they can be covered with the same or adifferent coating, such as a bioerodable polymer, as described below.

[0078] In an exemplary embodiment, the surface character of the coatingmaterial is altered or manipulated by including certain additives ormodifiers in the coating material during its manufacture. A method ofpreparing surface-functionalized polymeric materials by this method isset forth in, for example, Caldwell, supra. In the Caldwell method,additives or modifiers are combined with the polymeric material duringits manufacture. These additives or modifiers include compounds thathave affinity sites, compounds that facilitate the controlled release ofagents from the polymeric material into the surrounding environment,catalysts, compounds that promote adhesion between the bioactivematerials and the coating material and compounds that alter the surfacechemistry of the coating material.

[0079] As used herein, the term “affinity site” refers to a site on thepolymer that interacts with a complementary site on a biologicallyactive agent, or on the exterior surface of the structure to which thematrix is applied.

[0080] Affinity sites contemplated in the practice of the presentinvention include such functional groups as hydroxyl, carboxyl,carboxylic acid, amine groups, hydrophobic groups, inclusion moieties(e.g., cyclodextrin, complexing agents), biomolecules (e.g. antibodies,haptens, saccharides, peptides) and the like, that promote physicaland/or chemical interaction with the bioactive agent or tissue. In thepresent embodiment, the affinity site interacts with a bioactive agentor tissue by non-covalent means. The particular compound employed as themodifier will depend on the chemical functionality of the biologicallyactive agent and/or the groups on the surface of a particular tissue.Appropriate functional groups for a particular purpose can readily bededuced by one of skill in the art.

[0081] In another preferred embodiment, the coating used in theinvention is a substantially flowable material that can be delivered toa site of insult by means of, for example, a catheter, needle or otherpercutaneous delivery device. Preferred embodiments of the substantiallyflowable material are those that cure to a substantially non-flowablecoating in vivo. Materials meeting these criteria include, for example,fibrin sealants, hydrophobic poly(hydroxy acids) and the like. Thesubstantially flowable material will generally include one or morebiologically active agents. The amount of a particular biologicallyactive material contained in the substantially flowable material variesdepending on a number of factors, including, for example, the activityof the agent and the tenaciousness with which the agent adheres to thedelivery matrix. .

[0082] In another preferred embodiment, the biologically active materialinteracts with a surfactant that adheres to the coating material.Presently preferred surfactants are selected from benzalkonium halidesand sterylalkonium halides. Other surfactants suitable for use in thepresent invention are known to those of skill in the art.

[0083] In a still further preferred embodiment, the bioactive materialinteracting with, and adhering to, the coating material is a taxane, ataxane derivative or a combination thereof.

[0084] E. Delivery Vehicle Formats

[0085] The present invention includes providing a coating layer over asite of insult to an internal structure. In a preferred embodiment, thesite of insult is at least partially covered with a coating controllingthe release of at least one biologically active material dispersedthroughout the coating. Other preferred coatings comprise a reservoircomponent formed by, or entrapped within, the coating. The reservoircontains the biologically active material and, preferably, controls itsrelease properties. The reservoir can be a monolithic structure or itcan be formed by smaller structures dispersed in the coating (e.g.,microspheres).

[0086] The coating can take a number of forms. For example, usefulcoatings can be in the form of foams, gels, suspensions, microcapsules,solid polymeric materials and fibrous or porous structures. The coatingcan be multilayered with one or more of the layers including abiologically active material. Moreover, the coating can be layered on acomponent impregnated with a biologically active agent. Alternatively,the bioactive agent can be dispersed in one or more components orregions of the coating. Many materials that are appropriate for use ascoatings in the present methods are known in the art and both naturaland synthetic coatings are useful in practicing the present invention.

[0087] 1. Selection of Coating Materials

[0088] Suitable polymers that can be used as coatings in the presentinvention include, but are not limited to, water-soluble andwater-insoluble, biodegradable and nonbiodegradable polymers. Thecoatings of use in the present invention are preferably biodegradable,or more preferably bioerodable. The coatings are preferably sufficientlyporous, or capable of becoming sufficiently porous, to permit efflux ofthe biologically active molecules from the coating. The coatings arealso preferably sufficiently non-inflammatory and are biocompatible sothat inflammatory responses do not prevent the delivery of thebiologically active molecules to the tissue. It is advantageous if thecoating also provides at least partial protection of the biologicallyactive molecules from the adverse effects of proteases, hydrolases,nucleases and other relevant degradative species. In addition, it isadvantageous for the coating to produce controlled, sustained deliveryof the biologically active agent.

[0089] Many polymers can be utilized to form the coating. A coating canbe, for example, a gel, such as a hydrogel, organogel orthermoreversible gel. Other useful polymer types include, but are notlimited to, thermoplastics and films. Moreover, the coating can comprisea homopolymer, copolymer or a blend of these polymer types. The coatingcan also include a drug-loaded microparticle dispersed within acomponent of the coating, which serves as a dispersant for themicroparticles. Microparticles include, for example, microspheres,microcapsules and liposomes.

[0090] The coating matrix can serve to immobilize the microparticles ata particular site, enhancing targeted delivery of the encapsulatedbiologically active molecules. Rapidly bioerodible polymers such aspolylactide-co-glycolide, polyanhydrides, and polyorthoesters, whosecarboxylic groups are exposed on the surface are useful in the coatingsof use in the invention. In addition, polymers containing labile bonds,such as polyesters, are well known for their hydrolytic reactivity. Thehydrolytic degradation rates of the coatings can generally be altered bysimple changes in the polymer backbone.

[0091] The coating can be made up of natural and/or synthetic polymericmaterials. Representative natural polymers of use as coatings in thepresent invention include, but are not limited to, proteins, such aszein, modified zein, casein, gelatin, gluten, serum albumin, orcollagen, and polysaccharides, such as cellulose, dextrans, andpolyhyaluronic acid. Also of use in practicing the present invention arematerials, such as collagen and gelatin, which have been widely used onimplantable devices, such as textile grafts (see, for example, Hoffman,et al., U.S. Pat. Nos. 4,842,575, which issued on Jun. 27, 1989 and5,034,265, which issued on Jul. 23, 1991), but which have not beenutilized as components of adherent coatings for periadventitial deliveryof bioactive agents, such as those preventing or retarding thedevelopment if intimal hyperpalsia. Hydrogel or sol-gel mixtures ofpolysaccharides are also known. Furthermore, fibrin, an insolubleprotein formed during the blood clotting process, has also been used asa sealant for porous implantable devices (see, for example, Sawhey etal., U.S. Pat. No. 5,900,245, issued May 4, 1999). Useful fibrin sealantcompositions are disclosed in, for example, Edwardson et al., U.S. Pat.No. 5,770,194, which issued on Jun. 23, 1998 and U.S. Pat. No.5,739,288, which issued on Apr. 14, 1998. These and other naturallybased agents, alone or in combination, can be used as a coating inpracticing the present invention.

[0092] Representative synthetic polymers include, but are not limitedto, polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

[0093] Synthetically modified natural polymers include, but are notlimited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, and nitrocelluloses. Particularly preferred members ofthe broad classes of synthetically modified natural polymers include,but are not limited to, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxymethyl cellulose,cellulose triacetate, cellulose sulfate sodium salt, and polymers ofacrylic and methacrylic esters and alginic acid.

[0094] These and the other polymers discussed herein can be readilyobtained from commercial sources such as Sigma Chemical Co. (St. Louis,Mo.), Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

[0095] 2. Biodegradable and Bioresorbable Coating Materials

[0096] Coating compositions preferably have intrinsic and controllablebiodegradability, so that they persist for about a week to about sixmonths. The coatings are also preferably biocompatible, non-toxic,contain no significantly toxic monomers and degrade into non-toxiccomponents. Moreover, preferred coatings are chemically compatible withthe substances to be delivered, and tend not to denature the activesubstance. Still further preferred coatings are, or become, sufficientlyporous to allow the incorporation of biologically active molecules andtheir subsequent liberation from the coating by diffusion, erosion or acombination thereof. The coatings should also remain at the site ofapplication by adherence or by geometric factors, such as by beingformed in place or softened and subsequently molded or formed intofabrics, wraps, gauzes, particles (e.g., microparticles), and the like.Types of monomers, macromers, and polymers that can be used aredescribed in more detail below.

[0097] Representative biodegradable polymers include, but are notlimited to, polylactides, polyglycolides and copolymers thereof,poly(ethylene terephthalate), poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), poly(lactide-co-glycolide),polyanhydrides, polyorthoesters, blends and copolymers thereof. Ofparticular use are compositions that form gels, such as those includingcollagen, pluronics and the like.

[0098] Still further preferred coatings are water-insoluble materialsthat comprise within at least a portion of their structure, abioresorbable molecule. An example of such a coating is one thatincludes a water-insoluble copolymer, which has a bioresorbable region,a hydrophilic region and a plurality of crosslinkable functional groupsper polymer chain.

[0099] For purposes of the present invention, “water-insolublematerials” includes copolymers that are substantially insoluble in wateror water-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thecopolymer molecule, as a whole, does not by any substantial measuredissolve in water or water-containing environments.

[0100] For purposes of the present invention, the term “bioresorbablemolecule” includes a region that is capable of being metabolized orbroken down and resorbed and/or eliminated through normal excretoryroutes by the body. Such metabolites or break down products arepreferably substantially non-toxic to the body.

[0101] The bioresorbable region is preferably hydrophobic. In anotherembodiment, however, the bioresorbable region may be designed to behydrophilic so long as the copolymer composition as a whole is notrendered water-soluble. Thus, the bioresorbable region is designed basedon the preference that the copolymer, as a whole, remainswater-insoluble. Accordingly, the relative properties, i.e., the kindsof functional groups contained by, and the relative proportions of thebioresorbable region, and the hydrophilic region are selected to ensurethat useful bioresorbable compositions remain water-insoluble.

[0102] Exemplary resorbable coatings include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

[0103] Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly (amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebiosresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

[0104] In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

[0105] Higher order copolymers can also be used as coatings in themethods of the present invention. For example, Casey et al., U.S. Pat.No. 4,438,253, which issued on Mar. 20, 1984, discloses tri-blockcopolymers produced from the transesterification of poly(glycolic acid)and an hydroxyl-ended poly(alkylene glycol). Such compositions aredisclosed for use as resorbable monofilament sutures. The flexibility ofsuch compositions is controlled by the incorporation of an aromaticorthocarbonate, such as tetra-p-tolyl orthocarbonate into the copolymerstructure.

[0106] Other coatings based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha difunctional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

[0107] The monomers, polymers and copolymers of use in the presentinvention preferably form a stable aqueous emulsion, and more preferablya flowable liquid. The relative proportions or ratios of thebioresorbable and hydrophilic regions, respectively are preferablyselected to render the block copolymer composition water-insoluble.Furthermore, these compositions are preferably sufficiently hydrophilicto form a hydrogel in aqueous environments when crosslinked.

[0108] The specific ratio of the two regions of the block copolymercomposition for use as coatings in the present invention will varydepending upon the intended application and will be affected by thedesired physical properties of the implantable coating, the site ofimplantation, as well as other factors. For example, the composition ofthe present invention will preferably remain substantiallywater-insoluble when the ratio of the water-insoluble region to thehydrophilic region is from about 10:1 to about 1:1, on a percent byweight basis.

[0109] Preferred bioresorbable regions of coatings useful in the presentinvention can be designed to be hydrolytically and/or enzymaticallycleavable. For purposes of the present invention, “hydrolyticallycleavable” refers to the susceptibility of the copolymer, especially thebioresorbable region, to hydrolysis in water or a water-containingenvironment. Similarly, “enzymatically cleavable” as used herein refersto the susceptibility of the copolymer, especially the bioresorbableregion, to cleavage by endogenous or exogenous enzymes.

[0110] As set forth above, the preferred composition also includes ahydrophilic region. Although the present composition contains ahydrophilic region, in preferred coatings, this region is designedand/or selected so that the composition as a whole, remainssubstantially water-insoluble.

[0111] When placed within the body, the hydrophilic region can beprocessed into excretable and/or metabolizable fragments. Thus, thehydrophilic region can include, for example, polyethers, polyalkyleneoxides, polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol),poly(alkyl oxazolines), polysaccharides, carbohydrates, peptides,proteins and copolymers and mixtures thereof. Furthermore, thehydrophilic region can also be, for example, a poly(alkylene) oxide.Such poly(alkylene) oxides can include, for example, poly(ethylene)oxide, poly(propylene) oxide and mixtures and copolymers thereof.

[0112] Concerning the disposition of the biologically active agent inthe coating, substantially any combination of bioactive compound andcoating that is of use in achieving the object of the present inventionis contemplated by this invention. In a preferred embodiment, thebioactive material is dispersed in a resorbable coating that impartscontrolled release properties to the biologically active agent. Thecontrolled release properties can result from, for example, a resorbablepolymer that is cross-linked with a degradable cross-linking agent.Alternatively, the controlled release properties can arise from aresorbable polymer that incorporates the biologically active material ina network of pores formed during the cross-linking process or gelling.In another embodiment, the drug is loaded into microspheres, which arethemselves biodegradable and the microspheres are embedded in thecoating. Many other appropriate drug/coating formats will be apparent tothose of skill in the art.

[0113] In another preferred embodiment, an underlying polymericcomponent of a coating of use in the invention is first impregnated withthe biologically active material and a resorbable polymer is layeredonto the underlying component. In this embodiment, the impregnatedcomponent serves as a reservoir for the bioactive material, which candiffuse out through pores in a resorbable polymer network, through voidsin a polymer network created as a resorbable polymer degrades in vivo,or through a layer of a gel-like coating. Other controlled releaseformats utilizing a polymeric substrate, a bioactive agent and a coatingwill be apparent to those of skill in the art.

[0114] 3. Hydrogel-based Coatings

[0115] Also contemplated for use in the practice of the presentinvention as a coating component are hydrogels. Hydrogels are polymericmaterials that are capable of absorbing relatively large quantities ofwater. Examples of hydrogel forming compounds include, but are notlimited to, polyacrylic acids, sodium carboxymethylcellulose, polyvinylalcohol, polyvinyl pyrrolidine, gelatin, carrageenan and otherpolysaccharides, hydroxyethylenemethacrylic acid (HEMA), as well asderivatives thereof, and the like. Hydrogels can be produced that arestable, biodegradable and bioresorbable. Moreover, hydrogel compositionscan include subunits that exhibit one or more of these properties.

[0116] Bio-compatible hydrogel compositions whose integrity can becontrolled through crosslinking are known and are presently preferredfor use in the methods of the invention. For example, Hubbell et al.,U.S. Pat. Nos. 5,410,016, which issued on Apr. 25, 1995 and 5,529,914,which issued on Jun. 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

[0117] In a preferred embodiment, the bioactive material is dispersed ina hydrogel that is cross-linked to a degree sufficient to impartcontrolled release properties to the biologically active agent. Thecontrolled release properties can result from, for example, a hydrogelthat is cross-linked with a degradable cross-linking agent.Alternatively, the controlled release properties can arise from ahydrogel that incorporates the biologically active material in a networkof pores formed during the cross-linking process.

[0118] In another preferred embodiment, the gel is a thermoreversiblegel. Thermoreversible gels including components, such as pluronics,collagen, gelatin, hyalouronic acid, polysaccharides, polyurethanehydrogel, polyurethane-urea hydrogel and combinations thereof arepresently preferred.

[0119] In yet another preferred embodiment, a component of the coatingis first impregnated with the biologically active material and thehydrogel is layered onto the impregnated coating component. In thisembodiment, the impregnated coating component serves as a reservoir forthe bioactive material or agent, which can diffuse out through pores inthe hydrogel network or, alternatively, can diffuse out through voids inthe network created as the hydrogel degrades in vivo (see, for example,Ding et al., U.S. Pat. No. 5,879,697, issued Mar. 9, 1999; and Ding etal., U.S. Pat. No. 5,837,313, issued Nov. 17, 1998). Other controlledrelease formats utilizing a polymeric substrate, a bioactive agent and ahydrogel will be apparent to those of skill in the art.

[0120] As set forth above, useful coatings of the present invention canalso include a plurality of crosslinkable functional groups. Anycrosslinkable functional group can be incorporated into thesecompositions so long as it permits or facilitates the formation of ahydrogel. Preferably, the crosslinkable functional groups of the presentinvention are olefinically unsaturated groups. Suitable olefinicallyunsaturated functional groups include without limitation, for example,acrylates, methacrylates, butenates, maleates, allyl ethers, allylthioesters and N-allyl carbamates. Preferably, the crosslinking agent isa free radical initiator, such as for example, 2,2′-azobis(N,N′dimethyleneisobutyramidine) dihydrochloride.

[0121] The crosslinkable functional groups can be present at any pointalong the polymer chain of the present composition so long as theirlocation does not interfere with the intended function thereof.Furthermore, the crosslinkable functional groups can be present in thepolymer chain of the present invention in numbers greater than two, solong as the intended function of the present composition is notcompromised.

[0122] An example of a coating having the above-recited characteristicsis found in, for example, Loomis, U.S. Pat. No.5,854,382, issued Dec.29, 1998. This coating is exemplary of the types of coatings that can beused in the invention.

[0123] Also contemplated by the present invention is the use of coatingsthat are capable of promoting the release of an agent from the coating.For example, in a preferred embodiment, the bioactive material isdispersed throughout the hydrogel. As the hydrogel degrades byhydrolysis or enzymatic action, the bioactive material is released.Alternatively, the coating may promote the release of a biologicallyactive material by forming pores once the resulting article is placed ina particular environment (e.g., in vivo). In a preferred embodiment,these pores communicate with a reservoir containing the bioactivematerial. Other such coating components that promote the release of anagent from materials are known to those of skill in the art.

[0124] F. Fibrin Sealants

[0125] In a particularly preferred embodiment, the drug delivery vehicleused in the methods of the invention is a fibrin sealant. Fibrinsealants having substantially any composition are useful in the methodsof the present invention.

[0126] Fibrin sealants are biological adhesives whose effect imitatesthe final stages of coagulation, thereby resulting in a fibrin clot.Conventional fibrin sealants consist of concentrated human fibrinogen,bovine aprotinin and factor XIII, as the first component and bovinethrombin and calcium chloride as the second component. Application isgenerally carried out with a double-barreled syringe, which permitssimultaneous application of both components to the site where one wantsto form the fibrin clot. Aprotinin is a fibrinolytic inhibitor, whichcan be added to promote stability of fibrin sealants.

[0127] The fibrinogen component of the fibrin sealant can be preparedfrom pooled human plasma. The fibrinogen can be concentrated from thehuman plasma by cryoprecipitation and precipitation using variousreagents, e.g., polyethylene glycol, ether, ethanol, ammonium sulfate orglycine. For an excellent review of fibrin sealants, see, Brennan, BloodReviews 5:240-244 (1991); Gibble et al., Transfusion 30:741-747 (1990);Matras, Oral Maxillofac Sura. 43:605-611 (1985) and Lerner et al., J. ofSurgical Research 48:165-181 (1990).

[0128] Recently, there has also been an interest in the preparation offibrin sealants that utilize autologous fibrin. An autologous fibrinsealant is a fibrin sealant wherein the fibrinogen component of thefibrin sealant is extracted from the patient's own blood. The use of anautologous fibrin sealant is presently preferred because it eliminatesthe risk of transmission of blood-transmitted infections, e.g.,hepatitis B, non A, non B hepatitis and acquired immune deficiencysyndrome (AIDS), that could otherwise be present in the fibrinogencomponent extracted from pooled human plasma. See, Silberstein et al.,Transfusion 28:319-321 (1988); Laitakari et al., Laryngoscope 99:974-976(1989) and Dresdale et al., Ann. Thoracic Surgery 40:385-387 (1985).

[0129] Fibrin sealants useful in the methods of the invention canutilize crosslinked fibrin sealants, non-cross-linked fibrin sealantsand combinations thereof. Non-limiting examples of non-crosslinkedfibrin are non-crosslinked fibrin I, non-crosslinked fibrin II and desBB fibrin, with non-crosslinked fibrin I being preferred. Mixtures ofnon-crosslinked fibrin can be present. Also, for the purpose of thesubject invention “crosslinked fibrin” includes any form of fibrinresulting from the conversion of non-crosslinked fibrin to crosslinkedfibrin. Thus, the crosslinked fibrin, for example, resulting from theconversion of non-crosslinked fibrin I to crosslinked fibrin, can becrosslinked fibrin I and/or crosslinked fibrin II, depending on how theconversion step is carried out.

[0130] F. Microencapsulation of Bioactive Material

[0131] In another preferred embodiment, the biologically active materialis incorporated into a polymeric component by encapsulation in amicrocapsule. The microcapsule is preferably fabricated from a materialdifferent from that of the bulk of the coating matrix.

[0132] Preferred microcapsules are those which are fabricated from amaterial that undergoes erosion in the host, or those which arefabricated such that they allow the bioactive agent to diffuse out ofthe microcapsule. Such microcapsules can be used to provide for thecontrolled release of the encapsulated biologically active material fromthe microcapsules.

[0133] Numerous methods are known for preparing microparticles of anyparticular size range. In the various delivery vehicles of the presentinvention, the microparticle sizes may range from about 0.2 micron up toabout 100 microns. Synthetic methods for gel microparticles, or formicroparticles from molten materials, are known, and includepolymerization in emulsion, in sprayed drops, and in separated phases.For solid materials or preformed gels, known methods include wet or drymilling or grinding, pulverization, size separation by air jet, sieve,and the like.

[0134] Microparticles can be fabricated from different polymers using avariety of different methods known to those skilled in the art.Exemplary methods include those set forth below detailing thepreparation of polylactic acid and other microparticles.

[0135] Polylactic acid microparticles are preferably fabricated usingone of three methods: solvent evaporation, as described by Mathiowitz,et al., J. Scanning Microscopy 4:329 (1990); Beck, et al., Fertil.Steril. 31: 545 (1979); and Benita, et al., J. Pharm. Sci. 73: 1721(1984); hot-melt microencapsulation, as described by Mathiowitz, et al.,Reactive Polymers 6: 275 (1987); and spray drying. Exemplary methods forpreparing microencapsulated bioactive materials useful in practicing thepresent invention are set forth below.

[0136] 1. Solvent Evaporation

[0137] In this method, the microcapsule polymer is dissolved in avolatile organic solvent, such as methylene chloride. The drug (eithersoluble or dispersed as fine particles) is added to the solution, andthe mixture is suspended in an aqueous solution that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent has evaporated, leaving solidmicroparticles. The solution is loaded with a drug and suspended invigorously stirred distilled water containing poly(vinyl alcohol)(Sigma). After a period of stirring, the organic solvent evaporates fromthe polymer, and the resulting microparticles are washed with water anddried overnight in a lyophilizer. Microparticles with different sizes(1 - 1000 microns) and morphologies can be obtained by this method. Thismethod is useful for relatively stable polymers like polyesters andpolystyrene. Labile polymers such as polyanhydrides, may degrade duringthe fabrication process due to the presence of water. For thesepolymers, the following two methods, which are performed in completelyanhydrous organic solvents, are preferably used.

[0138] 2. Hot Melt Microencapsulation

[0139] In this method, the polymer is first melted and then mixed withthe solid particles of biologically active material that have preferablybeen sieved to less than 50 microns. The mixture is suspended in anon-miscible solvent (like silicon oil) and, with continuous stirring,heated to about 5° C. above the melting point of the polymer. Once theemulsion is stabilized, it is cooled until the polymer particlessolidify. The resulting microparticles are washed by decantation with asolvent such as petroleum ether to give a free-flowing powder.Microparticles with sizes ranging from about 1 to about 1000 microns areobtained with this method. The external surfaces of capsules preparedwith this technique are usually smooth and dense. This procedure ispreferably used to prepare microparticles made of polyesters andpolyanhydrides.

[0140] 3. Solvent Removal

[0141] This technique is preferred for polyanhydrides. In this method,the biologically active material is dispersed or dissolved in a solutionof the selected polymer in a volatile organic solvent like methylenechloride. This mixture is suspended by stirring in an organic oil (suchas silicon oil) to form an emulsion. Unlike solvent evaporation, thismethod can be used to make microparticles from polymers with highmelting points and different molecular weights. Microparticles thatrange from about 1 to about 300 microns can be obtained by thisprocedure. The external morphology of spheres produced with thistechnique is highly dependent on the type of polymer used.

[0142] 4. Spray-Drying

[0143] In this method, the polymer is dissolved in methylene chloride. Aknown amount of the active drug is suspended (insoluble drugs) orco-dissolved (soluble drugs) in the polymer solution. The solution orthe dispersion is then spray-dried. Microparticles ranging between about1 to about 10 microns are obtained with a morphology which depends onthe type of polymer used.

[0144] 5. Hydrogel Microparticles

[0145] In a preferred embodiment, the bioactive material is encapsulatedin microcapsules that comprise a sodium alginate envelope.

[0146] Microparticles made of gel-type polymers, such as alginate, arepreferably produced through traditional ionic gelation techniques. Thepolymers are first dissolved in an aqueous solution, mixed with bariumsulfate or some bioactive agent, and then extruded through amicrodroplet forming device, which in some instances employs a flow ofnitrogen gas to break off the droplet. A slowly stirred (approximately100-170 RPM) ionic hardening bath is positioned below the extrudingdevice to catch the forming microdroplets. The microparticles are leftto incubate in the bath for about twenty to thirty minutes in order toallow sufficient time for gelation to occur. Microparticle size iscontrolled by using various size extruders or varying either thenitrogen gas or polymer solution flow rates.

[0147] 6. Liposomes

[0148] Liposomes are commercially available from a variety of suppliers.Alternatively, liposomes can be prepared according to methods known tothose skilled in the art, for example, as described in Eppstein et al.,U.S. Pat. No. 4,522,811, which issued on Jun. 11, 1985. For example,liposome formulations may be prepared by dissolving appropriate lipid(s)(such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidylcholine, arachadoyl phosphatidyl choline, and cholesterol) in aninorganic solvent that is then evaporated, leaving behind a thin film ofdried lipid on the surface of the container. An aqueous solution of theactive compound or its pharmaceutically acceptable salt is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

[0149] The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, are of use in thepresent invention.

[0150] G. Bioactive Agent Release Rates

[0151] In another preferred embodiment, the methods of the inventioninclude the use of two or more populations of bioactive agents. Thepopulations are distinguished by, for example, having different rates ofrelease from the coating of the invention. Two or more different ratesof release can be obtained by, for example, incorporating one agentpopulation into the bulk coating and the other agent population intomicrocapsules embedded in the bulk coating. In another exemplaryembodiment, the two agents are encapsulated in microspheres havingdistinct release properties. For example, the first agent isencapsulated in a liposome and the second agent is encapsulated in analginate microsphere.

[0152] Other characteristics of the populations in addition to theirrelease rates can be varied as well. For example, the two populationscan consist of the same, or different agents. Moreover, theconcentrations of the two populations can differ from each other. Forexample, in certain applications it is desirable to have one agentreleased rapidly (e.g., an antibiotic) at a first concentration, while asecond agent is released more slowly at a second concentration (e.g., aninhibitor of tissue overgrowth). Furthermore when two or more distinctagents are used they can be distributed at two or more unique siteswithin the delivery vehicle.

[0153] In yet a further embodiment, the delivery vehicle of theinvention can control the release of two or more agents acting inconcert to achieve a biological effect. For example, vascularendothelial growth factor (VEGF)can initially be released from thematrix to recruit new vessels to a tissue. Some time prior to or afterthe exhaustion of the VEGF, a second agent, such as fibroblast growthfactor (Fgf) is released to stabilize the newly recruited vessels. Manyother such permutations of agent types, agent concentrations and agentrelease rates will be readily apparent to those of skill in the art.

[0154] H. Formation of a Polymer Matrix

[0155] In a preferred embodiment, a solid, flexible drug deliveryvehicle matrix is formed by dispensing a flowable polymer, or polymerprecursor, formulation onto the surface of a tissue which is surroundedby an aqueous medium. The formulation can be applied to a patient'stissues by any convenient technique. For example, the formulation can beapplied by brushing, spraying, extruding, dripping, injecting, orpainting. Spraying, via aerosolization is a preferred method ofadministration because it minimizes the amount of formulation applied tothe site of insult while maximizing uniformity. A thin, substantiallyuniform matrix, such as that formed by spraying, can also be called afilm. Typically, the film has a thickness of about 10 μm to about 10 mm,more preferably from about 20 μm to about 5 mm. Spraying is a preferredmethod for applying the polymer formulation to a large surface area,such as peritoneal sidewalls. In contrast, dripping may be preferred forapplying the polymer formulation to a small surface area, such as abowel resection or an anastomosis derived from a coronary artery bypass.

[0156] I. Characterization

[0157] Characterization of the bioactive agent, the coatings and thecombination of the bioactive agent and the coating can be performed atdifferent loadings of bioactive material to investigate coating andencapsulation properties and morphological characteristics of thecoatings and microparticles. Microparticle size can be measured byquasi-elastic light scattering (QELS), size-exclusion chromatography(SEC) and the like. Drug loading can be measured by dissolving thecoating or the microparticles into an appropriate solvent and assayingthe amount of biologically active molecules using one or moreart-recognized techniques. Useful techniques include, for example,spectroscopy (e.g., IR, NMR, UV/Vis, fluorescence, etc.), massspectrometry, elemental analysis, HPLC, HPLC coupled with one or morespectroscopic modalities, and other appropriate means.

[0158] J. Kits

[0159] The present invention also provides kits comprising the drugdelivery vehicles of the invention. By way of example, a fibrin sealantkit is described herein. The focus on fibrin sealant is intended to beillustrative and does not limit the scope of the invention.

[0160] The kit can contain as a first component a composition comprisingfibrin monomer and a buffer that is capable of solubilizing the fibrinmonomer or distilled water, depending on how the solubilization step wasperformed. The second component can optionally contain a source ofcalcium ions and/or thrombin. Alternatively, the first component can bea composition comprising noncrosslinked fibrin and the second componentcan be a source of calcium ions. If the source of fibrinogen utilized toprepare a composition comprising noncrosslinked fibrin is from cellcultures that secrete fibrinogen or recombinant fibrinogen, the firstcomponent can be a composition comprising noncrosslinked fibrin, thesecond component can be a source of calcium ions and a third componentis activated factor XIII.

[0161] In another embodiment, the kit comprises one or moreantihyperplastic agents, thrombin and a source of calcium ions. In thisembodiment, the fibrin is preferably derived from plasma removed fromthe patient into whom the delivery vehicle of the invention will beimplanted.

[0162] In addition to the drug delivery vehicle of the invention, thekit also contains directions concerning the use of the delivery vehiclesfor coating a site of insult on an external surface of an internalstructure. The kits can also optionally contain a device foradministering the vehicle in the method of the invention andbiologically active agents to be administered in conjunction with themethod of the invention. Other useful kit configurations will beapparent to those of skill in the art.

[0163] K. Methods of Treating Intimal Hyperplasia

[0164] Patients can be diagnosed for intimal hyperplasia using knownmethods, such as X-ray fluoroscopic examination of dye flowing through aparticular region of a blood vessel or other visual techniques, thepresence of symptoms such as pain, based on clinical judgment, or signsevidenced physical examination. Alternatively, it can be assumed thathyperplasia will arise due to performance of procedures such asangioplasty, arterial bypass graft, peripheral bypass surgery, or organtransplantation and the patient treated based on the assumption thatinjury or disease will inevitably arise.

[0165] In one embodiment, a coating comprising a bioactive agent isapplied to the site of insult during an open-field procedure. In anotherembodiment, the coating-drug composite are placed at the site of insultvia percutaneous means.

[0166] If intimal hyperplasia had been observed prior to implanting orwrapping the strips of matrices, the regression of hyperplasia istypically evidenced by a decrease in pain or other symptoms of decreasedblood flow, or through the use of imaging techniques. The decrease inhyperplasia or increase in flow rate through the injured vessel can bemonitored by the same methods used to initially diagnose the injury tothe vascular endothelium or blockage of the blood vessel.

EXAMPLES

[0167] 1.1 Materials

[0168] Paclitaxel® was obtained from Angiotech Pharmaceuticals, Inc.(Vancouver, Canada), and was supplied in proprietary micellar ordelayed-release microsphere formulations.

[0169] Fibrin Sealant, was supplied as Tisseel Fibrin Sealant kits,which were purchased from Baxter Healthcare Corp. These kits containedfibrinogen prepared from human plasma, and human thrombin.

[0170] Human FXIII was purchased from Enzyme Research Labs, So. Bend IN,and filter sterilized prior to use.

[0171] Reagents for all solutions were Reagent grade or better.

[0172] 1.2 Methods

[0173] 1.2a Method of Test Article and Vehicle Preparation

[0174] The fibrin polymer formulation, polymerized from a mixturecontaining a final concentration of 25-30 mg/ml fibrinogen, 5 IU humanfactor XIII, 50 IU human thrombin, ±Paclitaxel® was prepared by thefollowing method. 157 IU hFXIII was resuspended in 4 ml saline, mixedgently and sterilized by filtration and held until use on ice. Theresulting solution (2 ml) was added to each of two vials of the SealerProtein Concentrate (human) component of the Tisseel kit containingapproximately 190 mg fibrinogen/vial. The reconstituted sealant vialswere inverted to wet the pellet, and the vials held at 37° C. for 10minutes. An additional 2 ml of saline (±Paclitaxel®) was introduced intoeach vial, with continued gentle stirring. After visual inspection toinsure reconstitution of the Sealer Protein Concentrate, the contents ofthe vials were pooled and held at ambient temperature until use within 2hours. Formation of the fibrin polymer network was initiated at thevivarium by combination with a solution of human thrombin (100 IU/mL) in20 mM CaCl₂. The polymer was applied using the mixing devices suppliedwith the Tisseel kit.

[0175] Micellar Paclitaxel® was prepared as described as follows.Briefly, 4 mL sterile saline was added to one vial of Paclitaxel®reagent (11 mg/vial) and the vial incubated at 55° C. for 5 minutes. Thevial was mixed by vigorous vortexing for at least 2 minutes. The clearsolution (2 mL) was added to the Sealant Protein Concentrate asdescribed above.

[0176] Paclitaxel® microsphere solutions were prepared as follows. Eachvial of Paclitaxel® formulated in delayed-release microspheres wasreconstituted with 4 mL sterile saline, and 2 ml of this mixture wasadded per vial of Sealant Protein Concentrate.

[0177] 1.2b Animal Procedures

[0178] Dogs were purchased from Covance, a USDA approved vendor (10 maleand 2 female young adult animals) and quarantined as described in QCOPB600. Animals received standard laboratory diet proscribed in QCOP B618,with supplements provided at the discretion of a veterinarian; water wasavailable ad libitum.

[0179] Preoperative status of the animals was assessed by obtaining abaseline blood analysis including complete blood count and serumchemistries (IDEXX Veterinary Services, Inc. West Sacramento, Calif.)and by physical evaluation of the animals to monitor weight, bodytemperature, heart and respiratory rates. The animals were prepared forsurgery by insertion of an intravenous (iv) catheter placed in thecephalic vein.

[0180] Anesthesia was introduced through the cephalic iv catheter asdescribed in QCOP B803. An endotracheal tube was placed, and respiratorysupport provided. Anesthesia was maintained using a mixture ofisoflurane in oxygen. Lactated Ringers supplemented with a prophylacticadmixture of antibiotic (Cefazolin) was supplied intravenously.

[0181] Using aseptic surgical techniques, the femoral arteries and veinswere exposed and isolated. The veins were ligated and the proximal endtagged, and a segment harvested. The isolated vessel was flushed withsaline and trimmed to the desired size. A bilateral exposure of thecarotid arteries was performed.

[0182] The animal was heparinized (100 IU/ kg body weight), andanticoagulated at the surgeon's discretion with additional heparin. Thecarotid artery was cross-clamped and transected. An interpositionalgraft with end-to-side anastamoses of the femoral vein to the carotidartery was performed. Anastomoses were identified with a surgicalstaple. Where indicated, the test or control articles were applied to auniform thickness and allowed to harden. Grafts were placed in bothcarotid arteries, with the procedures done in series.

[0183] Balloon injury to the femoral arteries by three sequences ofinflation and removal of a 4 Fr. Fogarty catheter generated 5 cm lesionsin each femoral artery that were or were not treated with test/controlarticles. Injured areas were tagged with a staple. The insertion sitewas repaired and the animal was closed.

[0184] Post-operative care was performed as described in QCOP B809,along with prophylactic administration of antibiotics (Sulfamethoxaxoleand Trimethoprin); analgesics were supplied as described in QCOP B803. 7days post surgery, animals received 250 mg/day aspirin. The wound sitewas debrided and temperature, heart rate and respiratory rates weremonitored daily the week following surgery. Angiography of the carotidsites was performed following surgery and monthly thereafter.Intravascular ultrasound (IVUS) was used to examine the vein grafts in10 animals at the 12 week endpoint of the study.

[0185] At the termination of the experiment, the overall health of theanimals was monitored, including routine blood work. Animals wereanti-coagulated with heparin (300 IU/kg body weight) and angiography andIVUS measurements of treated vessels were taken. Animals wereanesthetized and euthanized as described in QCOP B803 and B621respectively.

[0186] Carotid arteries were exposed and the healing response wasevaluated. The grafts, including anastomoses, were fixed under pressurein situ, and were removed along with 4 cm of proximal and distal hostvessel. Femoral arteries were exposed and the healing response wasobserved. The femoral arteries were then fixed under pressure andremoved with distal and proximal host tissue. Femoral and carotidarteries were stored in 10% neutral-buffered formalin until analysis byhistology. Specimens for histological evaluation were processed andstained with Hematoxalin and Eosin and with Mallory's Trichrome by IDEXXVeterinary Services, West Sacramento, Calif.

[0187] 1.2c Data Analysis

[0188] 1.2c1 Carotid Vein Grafts

[0189] Image analysis was performed by a qualified vascular surgeon whoalso analyzed the data. REF Images from angiography were captured on aVHS tape. Digital images were captured from this tape and saved afterwhich they were evaluated using NIH Image. The lumenal width of thenative arterial segments proximal and distal to the vein grafts, and theproximal and distal anastomoses were measured. The percent stenoses ofthe anastomoses relative to the adjacent native arterial segment werereported.

[0190] 1.2c1a Femoral Arteries

[0191] Angiography of the femoral arteries was performed at thetermination of the experiment. Images were captured as described aboveand analyzed using NIH Image. Injured areas were identified byradio-opaque clips, and by anatomy. The lumenal widths of the arterialsegments were measured just distal to the arteriotomy, and in the mid-and distal portions of the injured segments. The percent stenoses of theanastomoses relative to the adjacent native arterial segment werereported. The mean and least values were reported.

[0192] Histology of the femoral arteries was analyzed with AdobePhotoshop. Briefly, (1) the lumen-vessel interface, (2) the internalelastic lamina, and (3) the external elastic lamina were traced ondigital images from H&E stained tissue with the greatest amount ofhyperplasia. The intimal area was determined by subtracting the areaencompassing (2) from (1). The medial area was determined by subtractingthe area encompassing (2) from (3). The area was measured as (1).

[0193] 1.3 Results

[0194] 1.3a Animal Studies

[0195] 1.3a1 Clinical Observations

[0196] The animals were assigned to test groups as shown in Table 1. Alldogs recovered uneventfully from surgery and anesthesia. In controlanimals, subcutaneous hematomas that resolved with time were observed inthe carotid (⅓) and femoral sites (⅓). Subcutaneous hematomas at thecarotid site were also observed in animals treated with the vehicle (⅔)or vehicle+Paclitaxel® ({fraction (4/8)}); these lesions resolved withtime. {fraction (2/8)} animals treated with Paclitaxel® had a hematomaat the femoral site, which also reduced in sized with time and healedwell. Except where noted, all incision sites remained dry.

[0197] Two animals were sacrificed prior to the termination of theexperiment. Animal 12 had uncontrolled bleeding from the left femoralsite 13 d post-surgery which was ligated and repaired. 28-30 d post-surgery, bleeding from the right carotid site in animal 12 that couldnot be managed by discontinuing aspirin and pressure led to earlytermination of this animal. Animal 11 was observed with a large swellingat the left carotid area and altered mental status 8 weeks post-surgeryand was euthanized and explanted. Interestingly, both animals weretreated with the delayed release formulation of Paclitaxel®.

[0198] 1.3b1 Angiography and IVUS

[0199] The stenosis and blood flow through grafts was assessed usingangiography. Angiography showed that all carotid grafts were patent atthe conclusion of surgery, and that the carotid grafts of the survivinganimals were patent at the termination of the study (12 weeks).Angiography at the termination of the experiment showed that bothfemoral arteries in {fraction (8/10)} animals were patent; one femoralartery in Animal 6 and Animal 10 appeared to be occluded at 12 weeks.

[0200] IVUS was used to evaluated carotid grafts in 10 animals at the 12week time point. Images showed patent vessels, with some suggestion ofintimal thickening in some samples. The images were not evaluatedfurther.

[0201] 1.3c Quantitative Analysis of Angiography and Histology

[0202] 1.3c1 Carotid Vein Grafts

[0203] Images were captured as described in section above, and wereevaluated by NIH Image. The lumenal width of native arterial segmentsproximal and distal to the graft and the proximal and distal anastomoseswere measured. The per cent stenoses of the anastomoses relative to thenative artery are reported in Table 2. There was one animal in theFibrin Sealant+microsphere Paclitaxel® due to mortality of two of theanimals; all other treatment groups represent data from three animals.The per cent stenoses of carotid grafts in animals treated withPaclitaxel® compared with untreated animals is shown in Table 3. TABLE 2% Stenoses of Carotid Grafts as Assessed by Angiography Proximal Numberof Anastomotic Distal Number Vessels Site Anastomotic Site Treatment ofDogs Evaluated (mean ± s.d) (mean ± s.d.) No. Treatment, 3 6 28.4 ± 14% 9.9 ± 13.4%  Control Fibrin vehicle 3 6 7.0 ± 80%* 6.9 ± 9.5%  Fibrinvehicle ± 3 6 7.9 ± 6.4%* 7.9 ± 6.4%* micellar Paclitaxel ® Fibrinvehicle ± 1 2 0.0 ± 0.0%* 0% microsphere Paclitaxel ®

[0204] TABLE 3 Per Cent Stenoses of Carotid Grafts ± Paclitaxel ® asAssessed by Angiography Proximal Number of Anastomotic Distal Treatment:Number Vessels Site Anastomotic Site Paclitaxel ® of Dogs Evaluated(mean ± s.d) (mean ± s.d.) No 6 12 15.9 ± 14.3%  8.4 ± 11.2%   Yes 4  85.9 ± 6.5%* 1.1 ± 2.2%**

[0205] 1.3c2 Balloon - injured Femoral Arteries: Angiography andHistology

[0206] Images from angiography were captured and analyzed as describedpreviously. The lumenal widths of the arterial segments were measuredimmediately distal to the arteriotomy, and in the mid- and distalportions of the injured segments. The data are expressed as a ratio ofthe measurement of injured : uninjured regions of the vessel and arereported in Tables 4 and 5. No significant differences were detectedbetween treatment groups using angiography to measure lumenal width.TABLE 4 Lumen Width of Balloon-Injured Segments Relative to NativeVessel Assessed by Angiography. Mean Num- Number of Lumenal LeastLumenal Treatment: ber of Vessels Width (mm) Width (mm) Paclitaxel ®Dogs Evaluated (Mean ± s.d.) (Mean ± s.d) No Treatment, 3 6 1.54 ± 0.181.23 ± 0.63 Control Fibrin vehicle 3 6 1.50 ± 0.3  1.41 ± 0.27 Fibrinvehicle ± 3 6 1.40 ± 0.25 1.32 ± 0.28 micellar Paclitaxel ® Fibrinvehicle ± 1 2 1.63 ± 0.09 1.55 ± 0.12 microsphere Paclitaxel ®

[0207] TABLE 5 Lumen Width of Balloon-Injured Segments Relative toNative Vessel ± Paclitaxel ® Mean Number of Lumenal Least LumenalTreatment: Number Vessels Width (mm) Width (mm) Paclitaxel ® of DogsEvaluated (Mean ± s.d.) (Mean ± s.d) No 6 12 1.46 ± 0.24 1.37 ± 0.27 Yes4  8 1.52 ± 0.24 1.32 ± 0.47

[0208] Histology of sections from areas spanning proximal and distalregions of the balloon-injured femorals and of native tissue proximal tothe arteriotomy were evaluated by morphometric analysis as describedabove. Measures of the lumen, intima, and media from areas with thegreatest hyperplasia are shown in Table 6, and this data was analyzedwith regard to presence or absence of Paclitaxel® and is presented inTable 7 and 8, respectively. TABLE 7 Measurement (Area) of Components ofFemoral Artery in Regions of Intimal Hyperplasia Number Intima: Numberof Vessels Media (mm²) Intima (mm²) Media Lumen (mm²) Treatment of DogsEvaluated (Mean ± s.d.) (Mean ± s.d.) Ratio (Mean ± s.d) No 3 6 2.31 ±0.50 0.92 ± 0.68  0.43 ± 0.37 3.09 ± 1.58   Treatment Fibnn 3 6 3.29 ±0.73* 2.34 ± 1.20* 0.7 ± 0.28 3.82 ± 1.02   vehicle Fibrin 3 6 3.59 ±0.87* 2.29 ± 0.86* 0.63 ± 0.17 4.91 ± 1.21* vehicle ± micellarPaclitaxel ® Fibrin 1 2 2.75 ± 0.34  0.96 ± 0.86  0.37 ± 0.36 5.19 ±0.57** vehicle ± microsphere Paclitaxel ®

[0209] TABLE 8 Measurement (Area) of Femoral Artery in Regions ofIntimal Hyperplasia ±Paclitaxel ® Number Intima: +HC, 36 Treatment:Number of Vessels Media (mm²) Intima (mm²) Media Lumen (mm²)Paclitaxel ® of Dogs Evaluated (Mean ± s.d.) (Mean ± s.d.) Ratio (Mean ±s.d) No 6 12 2.77 ± 0.77 1.63 ± 1.19 0.57 ± 0.34 3.45 ± 1.32 Yes 4  83.38 ± 0.84 1.95 ± 1.00 0.56 ±0.23 4.98 ± 1.05*

[0210]1.4 Discussion

[0211] 1.4a Carotid Grafts

[0212] Paclitaxel® limited stenosis at the proximal and distalanastomotic sites (Table 3, P=0.08 and 0.09, respectively), as assessedby angiography at the 12 week endpoint of the experiment. Analysis ofthe individual treatment groups revealed that the fibrin vehicle, in theabsence of paclitaxal, also limited stenosis at anastomotic sites (Table2). The data in Tale 2 suggest that healing of the vehicle and treatmentgroups were similar. This interpretation might be due to the smallsample size of the study. Consider the example of thevehicle+microsphere Paclitaxel® (slow release formulation) in which thegrafts in the surviving animal did not restenose. The trend seen in thisexample suggests that this formulation may limit stenosis.

[0213] 1.4b Femoral Arteries

[0214] Analysis of the data obtained by angiography suggested there wasno significant difference between control, vehicle and Paclitaxel®treatment groups ( Table 4 and 5). This contrasts with the data derivedby morphometric analysis of histology of the femoral sections with thelargest stenotic response. In this case, formulations with Paclitaxel®had a 44% larger lumen width (p</=0.06) in the absence of changes in theintimal: medial ratio (Table 7 and 8). The discrepancy between the dataderived by angiography and histology may reflect differences inmethodology of measurement, angiography being an in vivo assay with thedata measured in one-dimension, while histologic procedures aremulti-step, post-mortem procedures quantified in 2 dimensions. Theseresults suggest that histology is the more sensitive assay for thisanimal model at 12 weeks.

[0215] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to included within the spirit and purview of thisapplication and are considered within the scope of the appended claims.All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method of preventing or reducing intimalhyperplasia at a site of insult to an internal structure in a subject,said method comprising: contacting an exterior surface of said internalstructure contiguous with said site of insult, with a drug deliveryvehicle comprising an intimal hyperplasia preventing agent, wherein saiddrug delivery vehicle is substantially flowable during application tosaid exterior surface and substantially adheres to said exterior surfaceof said internal structure; and said drug delivery vehicle releasingsaid intimal hyperplasia preventing agent in a time dependent manner,said releasing occurring in an amount effective to prevent or reducesaid intimal hyperplasia.
 2. The method according to claim 1, whereinsaid internal structure is a structure having a substantially circularcross-section.
 3. The method according to claim 2, wherein said internalstructure is a member selected from vascular system component, anintestinal system component, a urinary system component and combinationsthereof.
 4. The method according to claim 1, wherein said injury is asurgical injury.
 5. The method according to claim 4, wherein saidinternal structure is a vascular structure and said surgical procedureis a member selected from the group consisting of angioplasty, vascularreconstructive surgery, heart valve replacement, heart transplantationand combinations thereof.
 6. The method according to claim 4, whereinsaid surgical injury comprises placing a prosthesis at said site ofinsult on said internal structure.
 7. The method according to 6, whereinsaid prosthesis comprises a member selected from a stent, a graft, avalve and combinations thereof at said site of insult on said internalstructure.
 8. The method according to claim 6, wherein said exteriorsurface of said vascular structure contacted with said drug deliveryvehicle comprises both said prosthesis and said site of insult.
 9. Themethod according to claim 1, wherein said site of insult comprises ananastomosis.
 10. The method according to claim 9, wherein said exteriorsurface of said vascular structure contacted with said drug deliveryvehicle comprises said anastomosis.
 11. The method according to claim 1,wherein said intimal hyperplasia preventing agent is a member selectedfrom antithrombotics, antiinflammatories, corticosteroids,antimicrotubule agents, antisense oligonucleotides, antineoplaastics,antioxidants, antiplatelets, calcium channel blockers, converting enzymeinhibitors, cytokine inhibitors, growth factors, growth factorinhibitors, growth factor sequestering agents, fibrosis inhibitors,immunosuppressives, tissue factor inhibitor, smooth muscle inhibitors,sulfated proteoglycans, superoxide dismutase mimics, NO, NO precursorsand combinations thereof.
 12. The method according to claim 11, whereinsaid antithrombotic is a member selected from heparin, heparinderivatives hirudin, hirudin derivatives and combinations thereof. 13.The method according to claim 11, wherein said corticosteroid isdexamethasone, dexamethasone derivatives and combinations thereof. 14.The method according to claim 11, wherein said antimicrotubule agent isa member selected from taxane, taxane derivatives and combinationsthereof.
 15. The method according to claim 11, wherein said antiplateletagent or said fibrosis inhibitor is an inhibitor of collagen synthesis.16. The method according to claim 15, wherein said inhibitor of collagensynthesis is selected from halofuginore, halofuginore derivatives,GpII_(b)III_(a) and combinations thereof.
 17. The method according toclaim 1, wherein said drug delivery vehicle is a member selected frombioerodable vehicles, hydrogel vehicles, thermoreversible vehicles,bioresorbable vehicles and combinations thereof.
 18. The methodaccording to claim 17, wherein said vehicle comprises a member selectedfrom gels, foams, suspensions, microcapsules, solid polymeric supportsand fibrous structures.
 19. The method according to claim 17, whereinsaid vehicle comprises a bioresorbable component.
 20. The methodaccording to claim 19, wherein said bioresorbable component is insolublein water.
 21. The method according to claim 20, wherein saidbioresorbable component is hydrophobic.
 22. The method according toclaim 19, wherein said bioresorbable component is hydrolytically and/orenzymatically cleavable.
 23. The method according to claim 19, whereinsaid bioresorbable component is selected from the group consisting ofpoly(esters), poly(hydroxy acids), poly(lactones), poly(amides),poly(ester-amides), poly (amino acids), poly(anhydrides),poly(orthoesters), poly(carbonates), poly(phosphazines),poly(phosphoesters), poly(thioesters), polysaccharides and mixturesthereof.
 24. The method according to claim 23, wherein saidbioresorbable component is a poly(hydroxy) acid.
 25. The methodaccording to claim 24, wherein said poly(hydroxy) acid comprises amaterial selected from the group consisting of poly(lactic) acid,poly(glycolic) acid, poly(caproic) acid, poly(butyric) acid,poly(valeric) acid and copolymers and mixtures thereof.
 26. The methodaccording to claim 17, wherein said vehicle forms a member selected fromexcretable fragments, metabolizable fragments and combinations thereof.27. The method according to claim 18, wherein said gel is athermoreversible gel.
 28. The method according to claim 27, wherein saidgel comprises a member selected from pluronics, fibrin sealants,albumin, collagen, gelatin, hydroxypropylmethylcellulose, organicpolymers, polyethylene oxide, hyalouronic acid, polysaccharides andcombinations thereof.
 29. The method according to claim 28, wherein saidgel comprises a member selected from polyurethane hydrogel andpolyurethane-urea hydrogel.
 30. The method according to claim 17,wherein said drug delivery vehicle comprises a member selected fromfibrin, fibronectin, thrombin and combinations thereof.
 31. The heartvalve according to claim 1, comprising a first population of bioactivematerial having a first release rate from said heart valve, and a secondbioactive material having a second release rate from said heart valve.32. The heart valve according to claim 31, wherein said first bioactivematerial and said second bioactive material are the same material. 33.The heart valve according to claim 31, wherein said first bioactivematerial and said second bioactive material are different materials. 34.The heart valve according to claim 31, wherein said first bioactivematerial is encapsulated in a microcapsule and said second bioactivematerial is admixed in a coating comprising said microcapsule.
 35. Amethod of preventing or reducing intimal hyperplasia at a site of insultto a vascular structure in a subject, wherein said insult is a memberselected from the group consisting of angioplasty, vascularreconstructive surgery and combinations thereof, said method comprising:contacting an exterior surface of said vascular structure contiguouswith said site of insult, with a drug delivery vehicle comprising anintimal hyperplasia preventing agent, wherein said drug delivery vehicleis substantially flowable during application to said exterior surfaceand substantially adheres to said exterior surface of said internalstructure; and said drug delivery vehicle releases said intimalhyperplasia preventing agent in a time dependent manner, said releaseoccurring in an amount effective to prevent or reduce said intimalhyperplasia.
 36. The method according to claim 35, wherein said vascularreconstructive surgery comprises placing a member selected from a stent,a graft and combinations thereof at the site of insult.
 37. The methodaccording to claim 36, wherein said exterior surface of said vascularstructure contacted with said drug delivery vehicle comprises a memberselected from both said stent and said site of insult, both said graftand said site of insult and combinations thereof.
 38. The methodaccording to claim 35, wherein said site of insult comprises ananastomosis.
 39. The method according to claim 38, wherein said exteriorsurface of said exterior surface of said vascular structure contactedwith said drug delivery vehicle comprises said anastomosis.
 40. Themethod according to claim 35, wherein said intimal hyperplasiapreventing agent is a member selected from antithrombotics,antiinflammatories, corticosteroids, antimicrotubule agents, antisenseoligonucleotides, antineoplaastics, antioxidants, antiplatelets, calciumchannel blockers, converting enzyme inhibitors, cytokine inhibitors,growth factors, growth factor inhibitors, growth factor sequesteringagents, fibrosis inhibitors, immunosuppressives, tissue factorinhibitor, smooth muscle inhibitors, sulfated proteoglycans, superoxidedismutase mimics, NO, NO precursors and combinations thereof.
 41. Themethod according to claim 40, wherein said antithrombotic is a memberselected from heparin, heparin derivatives hirudin, hirudin derivativesand combinations thereof.
 42. The method according to claim 40, whereinsaid corticosteroid is dexamethasone, dexamethasone derivatives andcombinations thereof.
 43. The method according to claim 40, wherein saidantimicrotubule agent is a member selected from taxane, taxanederivatives and combinations thereof.
 44. The method according to claim40, wherein said antiplatelet agent or said fibrosis inhibitor is aninhibitor of collagen synthesis.
 45. The method according to claim 44,wherein said inhibitor of collagen synthesis is selected fromhalofuginore, halofaginore derivatives, GpII_(b)III_(a) and combinationsthereof.
 46. The method according to claim 35, wherein said drugdelivery vehicle is a member selected from bioerodable vehicles,hydrogel vehicles, thermoreversible vehicles, bioresorbable vehicles andcombinations thereof.
 47. The method according to claim 46, wherein saiddrug delivery vehicle comprises a member selected from fibrin,fibronectin, thrombin and combinations thereof.
 48. A method of treatinga disease state of an internal structure in a subject, said methodcomprising: surgically treating said disease state, thereby creating asurgical site; and contacting an exterior surface of said internalstructure contiguous with said surgical site, with a drug deliveryvehicle comprising an intimal hyperplasia preventing agent, wherein saiddrug delivery vehicle is substantially flowable during application tosaid exterior surface and substantially adheres to said exterior surfaceof said internal structure; and said drug delivery vehicle releases saidintimal hyperplasia preventing agent in a time dependent manner, saidrelease occurring in an amount effective to prevent or reduce saidintimal hyperplasia.
 49. The method according to claim 48, wherein saidinternal structure has a substantially circular cross-section.
 50. Themethod according to claim 49, wherein said structure is a memberselected from vascular system component, an intestinal system component,a urinary system component and combinations thereof.
 51. The methodaccording to claim 48, wherein said internal structure is a vascularstructure and said surgical procedure is a member selected from thegroup consisting of angioplasty, vascular reconstructive surgery andcombinations thereof.
 52. The method according to claim 51, wherein saidvascular reconstructive surgery comprises placing a prosthesis at saidsurgical site.
 53. The method according to 52, wherein said prosthesiscomprises a member selected from a stent, a graft, a valve andcombinations thereof at said site of insult on said internal structure.54. The method according to claim 52, wherein said exterior surface ofsaid vascular structure contacted with said drug delivery vehiclecomprises a member selected from both said stent and said site ofinsult, both said graft and said site of insult and combinationsthereof.
 55. The method according to claim 48, wherein said site ofinsult comprises an anastomosis.
 56. The method according to claim 55,wherein said exterior surface of said vascular structure contacted withsaid drug delivery vehicle comprises said anastomosis.
 57. The methodaccording to claim 48, wherein said intimal hyperplasia preventing agentis a member selected from antithrombotics, antiinflammatories,corticosteroids, antimicrotubule agents, antisense oligonucleotides,antineoplaastics, antioxidants, antiplatelets, calcium channel blockers,converting enzyme growth factor sequestering agents, cytokineinhibitors, growth factors, growth factor inhibitors, fibrosisinhibitors, immunosuppressives, tissue factor inhibitor, smooth muscleinhibitors, sulfated proteoglycans, superoxide dismutase mimics, NO, NOprecursors and combinations thereof.
 58. The method according to claim57, wherein said antithrombotic is a member selected from heparin,heparin derivatives hirudin, hirudin derivatives and combinationsthereof.
 59. The method according to claim 57, wherein saidcorticosteroid is dexamethasone, dexamethasone derivatives andcombinations thereof.
 60. The method according to claim 57, wherein saidantimicrotubule agent is a member selected from taxane, taxanederivatives and combinations thereof.
 61. The method according to claim57, wherein said antiplatelet agent or said fibrosis inhibitor is aninhibitor of collagen synthesis.
 62. The method according to claim 61,wherein said inhibitor of collagen synthesis is selected fromhalofuginore, halofuginore derivatives, GpII_(b)III_(a) and combinationsthereof.
 63. The method according to claim 48, wherein said drugdelivery vehicle is a member selected from bioerodable vehicles,hydrogel vehicles, thermoreversible vehicles, bioresorbable vehicles andcombinations thereof.
 64. The method according to claim 63, wherein saiddrug delivery vehicle comprises a member selected from fibrin,fibronectin, thrombin and combinations thereof.
 65. The method accordingto claim 48, wherein said disease is a member selected from peripheralvascular disease, coronary artery disease, cardiac disease, hyperplasticdisease.
 66. A kit comprising a bioadhesive drug delivery vehiclecomprising: (a) a biologically active agent preventing or reducingintimal hyperplasia; and (b) a set of instructions explaining the use ofsaid drug delivery vehicle.